The long-term goal of our work is to determine the mechanisms by which T lymphocytes are activated by very small numbers of MHC-peptide complexes on the surface of antigen presenting cells. This sensitivity determines the threshold for processes such as negative and positive selection as well as activation of mature T cells and thus may play an important role in tolerance and immunity. Work from our lab and others has shown that antigen receptors, adhesion molecules and cytoskeletal dynamics collaborate to form a highly ordered immunological synapse, which sustains signaling by unknown mechanisms. We have observed that the organization and dynamics of T cell antigen receptors in the immunological synapse depends upon which adhesion systems are engaged. A prominent feature of the immunological synapse formed with different adhesion systems is small TCR clusters in the periphery of the contact area. In Aim 1 we will test the role of peripheral TCR clusters in sustained signaling and T cell activation. We will identify an optimal combination of ICAM-1, CD48, CD80 and MHC-peptide complexes in supported planar bilayers and will compare this to what is observed with a professional antigen presenting cell type, the dendritic cell (DC). In addition to LFA-1, CD2, CD28 and TCR, the co-receptor molecule CD4 plays an important role in setting T cell sensitivity to antigen. In Aim 2 we will test the importance of different putative interactions of CD4 with MHC class II, Lck, membrane domains, the TCR and itself in T cell sensitivity to antigen. The actin cytoskeleton has an essential role in T cell responses to antigen. Recent advances in understanding the actin cytoskeleton indicate that there are at least two different types of cortical actin cytoskeleton that can be distinguished by their dynamics and associated proteins. Cofilin, cortactin and Arp2/3 are associated with dynamic lamellipodia, which are sites of sensitive signal initiation. The lamella is directly behind the lamellipodium and contains actin filaments that are stabilized by tropomyosin and the actin- integrin adapter protein talin. In Aim 3 we will investigate the actin based structures in immunological synapses formed with different adhesion systems. Then we will use RNA interference and overexpression studies to manipulate the balance between the lamellipodium and lemella to determine which of these structures are most important for sensitive antigen recognition.